Title

Author

Defense Date

1999

Document Type

Thesis

Degree Name

Master of Science

Department

Physiology

First Advisor

Anton C. Schoolwerth

Abstract

In metabolic acidosis, the early increase in PEPCK mRNA and enzyme protein content contributes to the accelerated rates of ammonium and glucose formation. In situ hybridization demonstrated that expression of PEPCK was confined to medullary rays of rat kidney cortex in controls and spread throughout the cortex 10 hours following NH4Cl feeding (Am.J. Physiol., 267: F400, 1994). To identify the specific nephron segments expressing PEPCK in control and acidotic conditions, the mRNA for PEPCK along the nephron of the rat kidney was localized using the technique of reverse transcription and polymerase chain reaction (RT-PCR) in individual microdissected S1, S2 and S3 segments of the rat proximal tubule. Two-millimeter segments of tubule were permeabilized, the mRNA reverse transcribed using oligo-dT as a downstream primer and the eDNA product amplified by PCR (35 cycles). Primers specific for β-actin were used to confirm transfer of tubule, and only tubules positive for β-actin were amplified with primers specific for PEPCK. Both primers were designed to span at least one intron to avoid amplification of genomic DNA. The PCR products were detected using agarose gel electrophoresis and ethidium bromide staining. Verification of PCR product was performed by restriction enzyme digestion.

Under control conditions, the number of tubules expressing PEPCK mRNA as detected by RT-PCR was greatest in the S3 segment, moderate in the S2 segment, and least in the S1 segment of the proximal tubule. Ten hours after gavage feeding of 20- mmol/kg bodyweight NH4Cl, strong signals for PEPCK were detected in all three proximal tubule segments. These data demonstrate the ability of the rat kidney cortex to modulate the expression of PEPCK mRNA along the proximal tubule under physiological conditions, and to increase expression of PEPCK mRNA during metabolic acidosis by the recruitment of additional cells in the proximal nephron.